Biomimetic extracellular matrix nanofibers electrospun with calreticulin

Abstract

The present disclosure provides polymeric matrices comprising calreticulin (CRbT) and methods of producing and using such matrices. The polymeric matrices are useful in treatment of wounds (e.g., chronic diabetic wound).

1 . A polymeric matrix comprising calreticulin, or a functional fragment or derivative thereof. 
     
     
         2 . The polymeric matrix of  claim 1 , wherein the derivative of calreticulin is a recombinant protein comprising calreticulin or a functional fragment of calreticulin. 
     
     
         3 . The polymeric matrix of  claim 1 or 2 , wherein the functional fragment is N-, P- or C-domain of calreticulin. 
     
     
         4 . The polymeric matrix of any one of  claims 1-3 , wherein the polymeric matrix comprises a synthetic polymer, natural polymer, or a combination thereof. 
     
     
         5 . The polymeric matrix of  claim 4 , wherein the synthetic polymer comprises polycaprolactone (PCL), polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), poly(l-lactic acid)-co-poly(ε-caprolactone) (PLCL), polyphosphazene, poly-N-vinylpyrrolidone, polyglycolic acid, polydimethylsiloxane, poly(ethylene oxide)-poly(butylene terephthalate), nylon, polyvinyl alcohol (PVA), polyethylene glycol (PEG), or a combination thereof. 
     
     
         6 . The polymeric matrix of  claim 4 , wherein the natural polymer comprises collagen, chitosan, gelatin, hyaluronic acid, chondroitin sulfate, silk fibroin, elastin, tropoelastin, fibrin, fibrinogen, carboxymethyl cellulose, cellulose, decellularized tissue matrix, or a combination thereof. 
     
     
         7 . The polymeric matrix of  claim 4 , wherein the polymeric matrix comprises PCL and collagen. 
     
     
         8 . The polymeric matrix of  claim 6 or 7 , wherein the collagen comprises type I collagen (Col1), type II collagen (Col2), type III collagen (Col3), type IV collagen (Col4), type V collagen (Col5), type VII collagen (Col7), or a combination thereof. 
     
     
         9 . The polymeric matrix of any one of  claims 6-8 , wherein the collagen comprises type I collagen (Col1). 
     
     
         10 . The polymeric matrix of any one of claims  7 - 10 , wherein the polymeric matrix comprises PCL and collagen at a weight-to-weight ratio of about 1:10 to 10:1. 
     
     
         11 . The polymeric matrix of  claim 10 , wherein the polymeric matrix comprises PCL and collagen at a weight-to-weight ratio of about 3:1. 
     
     
         12 . The polymeric matrix of any one of  claims 1-11 , wherein the polymeric matrix is in the form of nanofibers, foams, sponges, nonwoven meshes, spheres, hydrogels, or 3D printed filament structures. 
     
     
         13 . The polymeric matrix of  claim 12 , wherein the polymeric matrix is in the form of nanofibers. 
     
     
         14 . The polymeric matrix of  claim 12 , wherein the nanofibers have a diameter of about 10-1000 nm 
     
     
         15 . The polymeric matrix of  claim 13 , wherein the nanofibers have a diameter of about 100-500 nm 
     
     
         16 . The polymeric matrix of  claim 14 , wherein the nanofibers have a diameter of about 330 nm. 
     
     
         17 . The polymeric matrix of any one of  claims 1-16 , wherein the polymeric matrix comprises a concentration of calreticulin at about 1 pg-100 mg/mL. 
     
     
         18 . The polymeric matrix of  claim 17 , wherein the polymeric matrix comprises a concentration of calreticulin at about  1 pg- 10  mg/mL. 
     
     
         19 . The polymeric matrix of  claim 17 or 18 , wherein the polymeric matrix comprises a concentration of calreticulin at about 100 ng/ml. 
     
     
         20 . The polymeric matrix of any one of  claims 1-19 , wherein the polymeric matrix further comprises an additional agent. 
     
     
         21 . The polymeric matrix of  claim 20 , wherein the polymeric matrix further comprises a cytokine, a growth factor, a glycosaminoglycan, a heat shock protein, a proteoglycan, a glycoprotein, syndecan, gelatin, or any mixtures thereof. 
     
     
         22 . The polymeric matrix of  claim 21 , wherein said glycosaminoglycan is hyaluronic acid. 
     
     
         23 . The polymeric matrix of  claim 21 , wherein said proteoglycan is perlecan or heparin sulfate. 
     
     
         24 . The polymeric matrix of  claim 21 , wherein said glycoprotein is fibronectin. 
     
     
         25 . The polymeric matrix of  claim 21 , wherein said growth factor is selected from the group consisting of a platelet-derived growth factor, vascular endothelial growth factor, fibroblast growth factor, epidermal growth factor, transforming growth factor-beta, and any mixtures thereof. 
     
     
         26 . The polymeric matrix of any one of  claims 1-25 , wherein the polymeric matrix has a three-dimensional structure. 
     
     
         27 . The polymeric matrix of any one of  claims 1-26 , wherein the polymeric matrix is produced by electrospinning. 
     
     
         28 . The polymeric matrix of any one of  claims 1-27 , wherein the polymeric matrix has one or more of the following characteristics:
 1) induces proliferation and/or migration of keratinocytes;   2) induces proliferation and/or migration of fibroblasts;   3) induces expression of CD68 in monocytes;   4) induces TGF-β1, fibronectin, collagen, laminin-5, p-FAK, integrin α5, and/or integrin β1 protein levels in fibroblasts;   5) promotes a polarized cell shape, which is the morphlogial phenotype of a motogenic cell replete with lamellipodia and filopodia;   6) induces elongated, oriented, and aligned cells on the CRT-NFs where CRT is presented at the basal side of the cell as well as the apical side;   7) induces dermal fibroblasts from the plantar foot of a non-healing wounds to adopt the phenotype of dermal fibroblasts from the plantar foot of a healing wound; and   8) induces tissue regeneration upon application to a mammal acute or chronic wound.   
     
     
         29 . A method of producing a polymeric matrix, comprising:
 c) mixing calreticulin, or a functional fragment or derivative thereof, within a polymeric solution; and   d) fabricating the polymeric matrix from the solution generated in step (a) using electrospinning.   
     
     
         30 . The method of  claim 29 , wherein the derivative of calreticulin is a recombinant protein comprising calreticulin or a functional fragment of calreticulin. 
     
     
         31 . The method of  claim 29 or 30 , wherein the functional fragment is N-, P- or C-domain of calreticulin. 
     
     
         32 . The method of any one of  claims 29-31 , wherein the calreticulin, or functional fragment or derivative thereof, is present in a solution comprising calreticulin, or functional fragment or derivative thereof, and a buffer. 
     
     
         33 . The method of  claim 32 , wherein the buffer comprises an organic amine and a metal halide salt at a pH from about 6 to about 8. 
     
     
         34 . The method of  claim 33 , wherein the organic amine is tromethamine. 
     
     
         35 . The method of  claim 33 , wherein the metal halide salt is CaCl 2 . 
     
     
         36 . The method of  claim 32 , wherein the buffer comprises saline, or PBS (phosphate buffered saline). 
     
     
         37 . The method of any one of  claims 29-36 , wherein the polymeric solution comprises a synthetic polymer, natural polymer, or a combination thereof. 
     
     
         38 . The method of  claim 37 , wherein the synthetic polymer comprises polycaprolactone (PCL), polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), poly(l-lactic acid)-co-poly(ε-caprolactone) (PLCL), polyphosphazene, poly-N-vinylpyrrolidone, polyglycolic acid, polydimethylsiloxane, poly(ethylene oxide)-poly(butylene terephthalate), nylon, polyvinyl alcohol (PVA), polyethylene glycol (PEG), or a combination thereof. 
     
     
         39 . The method of  claim 37 , wherein the natural polymer comprises collagen, chitosan, gelatin, hyaluronic acid, chondroitin sulfate, silk fibroin, elastin, tropoelastin, fibrin, fibrinogen, carboxymethyl cellulose, cellulose, decellularized tissue matrix, or a combination thereof. 
     
     
         40 . The method of  claim 37 , wherein the polymeric solution comprises PCL and collagen. 
     
     
         41 . The method of  claim 40 , wherein the collagen comprises type I collagen (Col1), type II collagen (Col2), type III collagen (Col3), type IV collagen (Col4), type V collagen (Col5), or a combination thereof. 
     
     
         42 . The method of any one of  claims 39-41 , wherein the collagen comprises type I collagen (Col1). 
     
     
         43 . The method of any one of  claims 39-42 , wherein the polymeric matrix comprises PCL and collagen at a weight-to-weight ratio of about 1:10 to 10:1. 
     
     
         44 . The method of  claim 43 , wherein the polymeric solution comprises PCL and collagen at a weight-to-weight ratio of about 3:1. 
     
     
         45 . The method of any one of  claims 29-44 , wherein the polymeric solution comprises a solvent. 
     
     
         46 . The method of  claim 45 , wherein the solvent is 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), trifluoroacetic acid, dichloromethane, or chloroform. 
     
     
         47 . The method of any one of  claims 29-46 , wherein the mixing step (a) is carried out at a temperature of about 0 to about 25° C. 
     
     
         48 . The method of any one of  claims 29-47 , wherein the mixing step (a) is carried out at a temperature of about 4 to about 10° C. 
     
     
         49 . The method of any one of  claims 29-47 , wherein the mixing step (a) is carried out at a temperature of about 4° C. 
     
     
         50 . The method of any one of  claims 29-49 , wherein the final solution generated in step (a) comprises PCL and the solvent at a weight-to-volume ratio of about 1-20%. 
     
     
         51 . The method of any one of  claims 29-50 , wherein the final solution generated in step (a) comprises PCL and the solvent at a weight-to-volume ratio of about 10%. 
     
     
         52 . The method of any one of  claims 29-51 , wherein the fabrication step (b) is carried out at a temperature of about 20° C. to about 30° C. 
     
     
         53 . The method of any one of  claims 29-52 , wherein the fabrication step (b) is carried out at a relative humidity of about 10% to about 60%. 
     
     
         54 . The method of any one of  claims 29-53 , wherein the electrospinning in step (b) is carried out using an electric field of about 0.5-20 kV/cm. 
     
     
         55 . The method of any one of  claims 29-54 , wherein the electrospinning in step (b) is carried out using an electric field of about 1 kV/cm. 
     
     
         56 . The method of any one of  claims 29-55 , wherein the polymeric matrix is collected at a distance of 2 mm-50 cm. 
     
     
         57 . The method of any one of  claims 29-56 , wherein step (a) further comprises mixing an additional agent within the polymeric solution. 
     
     
         58 . The method of  claim 57 , wherein the additional agent is a cytokine, a growth factor, a glycosaminoglycan, a heat shock protein, a proteoglycan, a glycoprotein, syndecan, gelatin or any mixtures thereof. 
     
     
         59 . The method of  claim 57 , wherein said glycosaminoglycan is hyaluronic acid. 
     
     
         60 . The method of  claim 57 , wherein said proteoglycan is perlecan or heparin sulfate. 
     
     
         61 . The method of  claim 21 , wherein said glycoprotein is fibronectin. 
     
     
         62 . The method of  claim 57 , wherein said growth factor is selected from the group consisting of a platelet-derived growth factor, vascular endothelial growth factor, fibroblast growth factor, epidermal growth factor, transforming growth factor-beta, and any mixtures thereof. 
     
     
         63 . A polymeric matrix produced by the method of any one of  claims 29-62 . 
     
     
         64 . A method for treating a wound in a subject in need thereof, said method comprises applying the polymeric matrix of any one of  claims 1-28 and 63  to the wound in the subject. 
     
     
         65 . The method of  claim 64 , wherein the polymeric matrix is applied topically or internally to the wound. 
     
     
         66 . The method of  claim 64 or 65 , wherein the wound is an acute wound or a chronic wound. 
     
     
         67 . The method of  claim 66 , wherein the acute wound is a burn, injury, or surgical intervention. 
     
     
         68 . The method of  claim 66 , wherein the chronic wound is a chronic diabetic wound, a venous or arterial stasis ulcer, a pressure ulcer, or an ulcer resulting from sickle cell disease (SCU). 
     
     
         69 . The method of  claim 68 , wherein the chronic diabetic wound is diabetic foot ulcer (DFU). 
     
     
         70 . The method of any one of  claims 64-69 , wherein the wound is a post-surgical wound or an internal wound. 
     
     
         71 . A method for promoting healing of a chronic diabetic wound in a subject in need thereof, said method comprises applying the polymeric matrix of any one of  claims 1-28 and 63  to the wound in the subject. 
     
     
         72 . The method of  claim 71 , wherein applying the polymeric matrix to the wound results in tissue regeneration in which neogenic epidermal appendage appear and scarring is reduced or eliminated. 
     
     
         73 . A method of inducing migration and/or proliferation of diabetic fibroblasts in a chronic diabetic wound in a subject in need thereof, said method comprises contacting the fibroblasts with the polymeric matrix of any one of  claims 1-28 and 63 . 
     
     
         74 . The method of any one of  claims 71-73 , wherein the diabetic wound is diabetic foot ulcer (DFU). 
     
     
         75 . The method of any one of  claims 71-74 , wherein the polymeric matrix is applied topically or internally to the wound. 
     
     
         76 . The method of any one of  claims 64-75 , further comprising administering a cytokine, a chemokine, a growth factor, a glycosaminoglycan, a heat shock protein, a proteoglycan, a glycoprotein, gelatin, syndecan or any mixtures thereof. 
     
     
         77 . The method of  claim 76 , wherein said glycosaminoglycan is hyaluronic acid. 
     
     
         78 . The method of  claim 76 , wherein said proteoglycan is perlecan or heparin sulfate. 
     
     
         79 . The method of  claim 76 , wherein said glycoprotein is fibronectin. 
     
     
         80 . The method of  claim 76 , wherein said growth factor is selected from the group consisting of a platelet-derived growth factor, vascular endothelial growth factor, fibroblast growth factor, epidermal growth factor, transforming growth factor-beta, and any mixtures thereof. 
     
     
         81 . The method of any one of  claims 64-80 , wherein the subject is mammal. 
     
     
         82 . The method of any one of  claims 64-81 , wherein the subject is human.